Abstract

Telomeres protect DNA ends of linear eukaryotic chromosomes from degradation and fusion, and ensure complete replication of the terminal DNA through recruitment of telomerase. The regulation of telomerase is a critical area of telomere research and includes cis regulation by the shelterin complex in mammals and fission yeast. We have identified a key component of this regulatory pathway as the SUMOylation [the covalent attachment of a small ubiquitin-like modifier (SUMO) to target proteins] of a shelterin subunit in fission yeast. SUMOylation is known to be involved in the negative regulation of telomere extension by telomerase; however, how SUMOylation limits the action of telomerase was unknown until now. We show that SUMOylation of the shelterin subunit TPP1 homolog in Schizosaccharomyces pombe (Tpz1) on lysine 242 is important for telomere length homeostasis. Furthermore, we establish that Tpz1 SUMOylation prevents telomerase accumulation at telomeres by promoting recruitment of Stn1-Ten1 to telomeres. Our findings provide major mechanistic insights into how the SUMOylation pathway collaborates with shelterin and Stn1-Ten1 complexes to regulate telomere length.

Tpz1 is SUMOylated at K242 in a manner dependent on the SUMO E3 ligase Pli1. (A) Schematic representation of Tpz1 functional domains. Pot1-, Poz1-, and Ccq1-interacting domains, as well as a predicted OB fold domain (OBD), are shown. The SUMOylation consensus site and position of the SUMOylated lysine (K242) are also depicted. CPBD, Ccq1/Poz1-binding domain; PBD, Pot1-binding domain. (B) Tpz1 is SUMOylated on K242. His6-3HA-tagged Pmt3-aa or His6-3HA-tagged Pmt3-gg was ectopically expressed from a pREP1-derived vector in tpz1-5Flag (WT) or tpz1-K242R-5Flag (K242R) cells and then purified with Ni-NTA under denatured conditions. Copurifying Tpz1 was detected by anti-Flag Western blot analysis. A band corresponding to SUMOylated Tpz1 is indicated by an arrow. (C) Tpz1 SUMOylation can be detected in cells expressing endogenous levels of Tpz1 and Pmt3. Tpz1-5Flag (WT) and Tpz1-K242R-5Flag (K242R) were immunoprecipitated by anti-Flag antibody from cells expressing endogenous WT Pmt3 only or coexpressing WT and GFP-tagged Pmt3, and detected by Western blot analysis using anti-Flag or anti-Pmt3 antibodies. (D) Tpz1 is SUMOylated primarily by SUMO E3 ligase Pli1. His6-3HA–tagged Pmt3-aa or His6-3HA–tagged Pmt3-gg was ectopically expressed from pREP1-derived vectors in fission yeast cells with indicated genotypes, and Tpz1-5Flag protein was detected by Western blot analysis. His6-3HA–tagged Pmt3-modified Tpz1-5Flag is indicated by an arrow. The asterisk indicates a nonspecific band.